The present invention relates to novel preparations of osteogenic factors, methods for their isolation and uses thereof (to repair bone defects). The preparations so isolated exhibit the ability to promote or stimulate the formation of bone at the site of their application. Bone is a highly specialized connective tissue with unique mechanical properties derived from its extensive matrix structure. A network of fibrous bundles composed of the protein, collagen, is presumed to provide the tension-resistant behavior of bone. In addition, other materials including proteoglycans, noncollagenous proteins, lipids and acidic proteins associated with a mineral phase consisting primarily of poorly crystallized hydroxyapatite are deposited in the extensive matrix architecture of bone. Bone tissue is continuously renewed, by a process referred to as remodeling, throughout the life of mammals. This physiologic process might serve to maintain the properties of a young tissue.
The processes of bone formation and renewal are carried out by specialized cells. Osteogenesis vis-a-vis morphogenesis and growth of bone is presumably carried out by the "osteoblasts" (bone-forming cells). Remodeling of bone is apparently brought about by an interplay between the activities of the bone-resorbing cells called "osteoclasts" and the bone-forming osteoblasts. The bony skeleton is thus not only an architectural structure with a mechanical function but also is a living tissue capable of growth, modeling, remodeling and repair. Since these processes are carried out by specialized living cells, chemical (pharmaceutical/hormonal), physical and physicochemical alterations can affect the quality, quantity and shaping of bone tissue.
A variety of pathological disorders as well as physical stress (for example, fracture) necessitate active formation of bone tissue at rates that are significantly higher than that which can be supported by the normal milieu of the body. It is thus of value to identify physiologically acceptable substances (hormones/pharmaceuticals/growth factors) that can induce the formation of bone at a predetermined site where such substances are applied, for example, by implantation. Such agents could either provide a permissive matrix structure for the deposition of bone-forming cells, or stimulate bone-forming cells, or induce the differentiation of appropriate progenitors of bone-forming cells.
The presence of proteinaceous and prostaglandin-like growth stimulators for osteoblasts has been examined, see reviews: Raisz, et al., New Engl. J. Med., 309(1), 29-35 (1983) and Raisz, et al., New Engl. J. Med., 309(2), 83-89 (1983).
The observation that a bone graft from the same individual or a compatible individual leads to the formation of new healthy bone at the site of the graft, led to the hypothesis that bone contains active proteins which promote local osteogenesis. Urist, et al. disclosed evidence that bone matrix-associated noncollagenous proteins can be isolated by dissociative treatment of demineralized bone powder and that this mixture of noncollagenous proteins contain the local osteoinductive capability which was designated by Urist (e.g., Science, 150, 893 (1965)) as bone morphogenetic activity.
A variety of osteogenic, cartilage-inducing and bone-inducing protein preparations have been described in the art. Urist, et al. and others have described various partially fractionated protein preparations with osteoinductive properties. These preparations are fractionated from the noncollagenous protein mixture extracted using different dissociative treatment of demineralized bone powder and subjecting the extract to various protein fractionation steps. Several such preparations have been characterized by different assays to determine their biological activities and by protein components identified using different standard protein analytical methods.
Urist, et al., Proc. Natl. Acad. Sci. (USA), 81, 371-375 (1984), discloses that bovine BMP has an apparent molecular weight of 18.5K daltons. The publication further discloses other bone derived proteins with apparent molecular weights of 17.5K and 17K, proteins with higher molecular weights of 34K, 24K and 22K and a protein with a lower molecular weight of 14K. The publication provided the N-terminal sequence for the 17.5K protein which had an unblocked amino terminus.
Urist, European Patent Application No. 212,474, discloses peptide fragments having molecular weights between about 4K and 7K comprising at least an active portion of the osteoinductive and immunoreactive domain of the 17.5K BMP molecule.
Wang, et al., Patent Cooperation Treaty Application No. WO 88/00205, discloses a bovine bone inductive factor which is isolated from demineralized bone powder by a procedure comprising a number of chromatographic and dialysis steps. The bone inductive factor so isolated was found to contain, as judged by a non-reducing SDS-PAGE analysis, one or more proteins having a molecular weight of approximately 28,000 to 30,000 daltons. Reducing SDS-PAGE analysis of the active protein(s) yielded two major bands having the mobility of proteins having molecular weights of 18,000 daltons and 20,000 daltons respectively. Wang, et al., discloses three bovine proteins designated BMP-1, BMP-2 and BMP-3 where BMP is bone morphogenetic protein and provides peptide sequences for the proteins. Wang, et al., also discloses the nucleotide sequences and amino acid sequences predicted thereby of four human proteins designated BMP-1, BMP-2 Class I, BMP-2 Class II and BMP-3.
Wozney, et al., Science, 242, 1528-1533 (1988), describes the nucleotide sequences and amino acid sequences predicted thereby of three human complementary DNA clones (designated BMP-1, BMP-2A and BMP-3) corresponding to three polypeptides present in an extract of bovine bone which is capable of inducing de novo bone formation. Recombinant human BMP-1, BMP-2A and BMP-3 proteins were said to be independently capable of inducing the formation of cartilage in vivo. The nucleotide sequence and derived amino acid sequence of a fourth complementary DNA clone (designated BMP-2B) is also described. The BMP-1, BMP-2A, BMP-2B and BMP-3 proteins of this publication appear to correspond, respectively, to the BMP-1, BMP-2 Class I, BMP-2 Class II and BMP-3 proteins.
Kubersampath, et al., Patent Cooperation Treaty Application No. WO 89/09787 claiming priority based on applications including U.S. Ser. No. 179,406 filed Apr. 8, 1988 and Oppermann, et al., Patent Cooperation Treaty Application No. WO 89/09788 claiming priority based on applications including U.S. Ser. No. 179,406 filed April 8, 1988 disclose nucleotide sequences and amino acid sequences predicted thereby of a human protein designated OP-1 and certain consensus nucleotide sequences and their amino acid sequences predicted thereby. These recombinant proteins are said to be independently capable of inducing the formation of bone in vivo.
Wang, et al., Proc. Natl. Acad. Sci. USA, 87, pp. 2220-2224 (1990), describe the nucleotide sequence and amino acid sequence predicted thereby of a human protein designated BMP-2A, corresponding to a polypeptide present in an extract of bovine bone which is capable of inducing de novo bone formation. Recombinant human BMP-2A protein is said to be independently capable of inducing the formation of bone in vivo.
Kubersampath, et al., J. Biol. Chem., 265, 13198-13205 (1990), describes a bovine bone-derived protein that induces bone formation. The bone-inductive protein was found to contain, as judged by a non-reducing SDS-PAGE analysis, a protein with a molecular weight of approximately 30,000 daltons. Reducing SDS-PAGE analysis yielded two major bands corresponding to molecular weights of 18,000 and 16,000 daltons. The 18,000-dalton subunit is the protein product of the bovine equivalent of the human OP-1 gene and the 16,000-dalton subunit is the protein product of the bovine equivalent of the human BMP-2A gene.
Celeste, et al., Proc. Natl. Acad. Sci. USA, 87, 9843-9847 (1990), describe the human protein sequences derived from the nucleotide sequence of six genes encoding proteins related to TGF-.beta.. These encoded proteins are designated BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 and BMP-7.